Research

RESEARCH FOCUS

The Paulino Lab is focused on elucidating the mechanism of action of membrane transporters (secondary- and primary-active) and channels on a molecular level. Although of high pharmacological relevance (> 60% of current drugs target membrane proteins), how their three-dimensional structure relates to their function and vice-versa is poorly understood and requires an interdisciplinary approach at the interface of biology, chemistry and physics. To address these questions, we employ cryo-electron microscopy and contrast our findings with comprehensive functional studies. Projects in our group are driven by the fundamental question of how membrane transporters work: how does the protein architecture translate into function; what is the exact mechanism of action during translocation of compounds from one side of the membrane to the other; how are they regulated; how is malfunction related to a disease? Over the past years, we have gained a particular interest in membrane transport proteins that fall out-of-the-box, challenging the conceptual boundaries present when classifying transport mechanisms into merely primary-active transporters, secondary-active transporters, or channels. It is becoming increasingly evident that in the course of evolution conserved protein architectures not only evolved from one another but can merge together to adapt to different environmental and cellular requirements.

Research topics

TMEM16

Groovy Channels & Scramblases

The TMEM16 family, which is exclusively found in eukaryotes, shows a remarkable dual functionality. It encompasses members functioning as calcium-activated chloride channels, as well as members functioning as calcium-activated lipid scramblases, which catalyze the exchange of phospholipids between the two membrane leaflets. The structures of the fungal lipid scramblase nhTMEM16 from Nectria haematococca and that of the chloride channel mTMEM16A from mouse revealed the architectural differences that underlie the functional diversity of the family. The calcium-bound active X-ray structure of nhTMEM16 revealed a large membrane-spanning hydrophilic furrow, termed catalytic ‘subunit cavity’, which would be exposed to the lipid bilayer. This cavity provides a pathway for the polar moieties of lipids across the membrane, whereas the apolar acyl chains remain embedded in the hydrophobic core of the bilayer, resembling a ‘credit-card’ mechanism. By contrast, the cryo-EM structures of the anion channel mTMEM16A revealed a closed subunit cavity, where transmembrane-helix rearrangements have sealed the membrane-accessible furrow, forming a protein-enclosed aqueous pore suitable for ion conduction. Our recent studies on the murine TMEM16F and the fungal nhTMEM16 lipid scramblases in detergent and in nanodiscs, both in presence and absence of Ca2+, have further shed light on their regulation by Ca2+ and their mechanism of action. In particular for nhTMEM16, we were able to capture distinct calcium-bound states, provide insights into conformational transitions under activating conditions. Although activated by a common mechanism, ion conduction and lipid scrambling appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state (alternating pore/cavity mechanism). The studies of nhTMEM16 in nanodiscs further reveal how the scramblase distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement. This work is done in collaboration with the group of Raimund Dutzler at the University of Zurich, Switzerland.

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ASCT2

Anticancer drug target with an elevator mechanism

Other projects have, apart from a mechanistic question, also a social-economical application. This is demonstrated in our studies of the human neutral amino-acid transporter ASCT2 from the SLCA1 family that also comprises the human excitatory amino acid transporters EAAT1-5 and the prokaryotic GltPhand GltTk. ASCT2 is the main source of glutamine uptake in human cells, which is strongly linked to cancer cell growth, poor patient survival and a new hot-target in cancer therapy. We were able to solve the first structures of the human ASCT2 in a substrate-bound inward-occluded state and more recently a substrate-free inward-open state of ASCT2. The latter represents the first structure of any SLCA1 in an inward-open state, and answers a long-lasting key mechanistic question. It was known that these transporters work like an elevator, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin 2 (HP2) gates the extracellular access to the substrate-binding site. However, it has remained unclear how substrate binding and release is gated on the cytoplasmic side. Strikingly, our data show that the same structural element (HP2) serves as a gate in the inward-facing as in the outward-facing state, revealing that SLC1A transporters work as one-gate elevators instead of two-gate elevators as previously assumed. This observation is of great fundamental interest, but also has potential implications for drug design. A prominent consequences of the one-gate elevator mechanism is that large protein movements take place in the cell membrane during transport. We were able to identify several unassigned densities near the gate and surrounding the scaffold domain, which may represent potential allosteric binding sites and guide the design of lipidic-inhibitors for anticancer therapy. This work is done in collaboration with the group of Dirk Slotboom at the University of Groningen, The Netherlands.

see:Garaeva AA, et al. Nat Comm (2019)Garaeva AA, et al. NSMB (2018)

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KdpFABC

A true chimera: a P-type ATPase hijacks a channel

P-type ATPases ubiquitously pump cations across biological membranes to maintain vital ion gradients. Among those, the chimeric K+ uptake system KdpFABC is unique. This complex is expressed under stress conditions, when the external K+ concentration is too low for ubiquitous K+-transporters to maintain the internal potassium concentration. KdpFABC is composed of four subunits, whereby the KdpA subunit resembles a K+-channel and the KdpB subunit is classified as a P-type ATPase (primary-active transporter). While ATP hydrolysis is accomplished by the P-type ATPase subunit KdpB, K+ has been assumed to be transported by the channel-like subunit KdpA. A first crystal structure uncovered its overall topology, suggesting such a spatial separation of energizing and transporting units. We were able to determine two additional structures of the 157 kDa, asymmetric complex in an E1 and E2 state, respectively. Unexpectedly, the new structures suggest a so far unprecedent transport mechanism through two half-channels along KdpA and KdpB. It units the alternating-access mechanism of actively pumping P-type ATPases with the high affinity and selectivity of K+-channels. This way, KdpFABC functions as a true chimeric complex, synergizing the best features of otherwise separately evolved transport mechanisms. This work is done in collaboration with the group of Inga Hänelt at the Goethe University in Frankfurt, Germany.

see:Stock et al., Nat Comm (2018)

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Upcoming soon

There are so many more things to explore in transporters and channels. So much still to understand. Stay tuned to see on what else we are working…